Microtubule-associated proteins characteristic of embryonic brain are found in the adult mammalian retina

We have used monoclonal antibodies against each of the major mammalian brain microtubule-associated proteins (MAPs), MAP1, MAP2, MAP3, MAP5, and tau, to study the timing of appearance and the cytological distribution of these proteins during the development of the rat retina. Western blots of adult rat retina reveal MAPs that are characteristic of embryonic brain, i.e., MAP5 and the low-molecular-weight forms of MAP2 (MAP2c) and tau (juvenile tau). At the onset of neuronal differentiation within the embryonic retina, MAP5, MAP3, MAP2c, and tau are found in the perikarya or extending axons of ganglion cells. High-molecular-weight MAP2, a dendrite marker, does not appear in the retina until the second day of postnatal development, when ganglion cell dendrites ramify within the inner plexiform layer. MAP1, which is characteristic of adult brain, does not appear in the retina until 1 week after birth, and is limited to ganglion cells and their processes. In the adult retina, MAP5 and MAP2c are concentrated within the inner segments and cell bodies of photosensitive cells, whereas tau is found in horizontal cells and more internal cell layers. Since photosensitive cells are unique among retinal neurons in their constant regeneration of their primary processes, the photoreceptive outer segments, both MAP5 and MAP2c appear not only to be involved in events associated with the embryonic differentiation and growth of neurites, but also in process regeneration in adult neurons that maintain some embryonic characteristics.     

Developmental regulation of two microtubule-associated proteins (MAP2 and MAP5) in the embryonic avian retina

Previous studies with the mammalian brain have shown that the expression of a number of neuronal microtubule-associated proteins (MAPs) is developmentally regulated. For example, the low-molecular-weight form of MAP2 (MAP2c) is abundant in neonatal rat brains and is less abundant in adults. Similarly, MAP5 levels decrease during postnatal development. Using monoclonal antibodies, we have followed the time of first appearance, cellular distribution, and molecular form of MAP2 and MAP5 during the morphogenesis of the quail retina. MAP2 first appears in ganglion cell bodies and in the axons of the optic fibre layer (OFL) at embryonic day 4 (E4). Anti-MAP2 staining remains restricted to these sites until E10, when staining appears in the inner plexiform layer (IPL). At E14, one day before hatching, anti-MAP2 staining is found in three broad laminae in the IPL, as well as in photosensitive cells. MAP5 is present in ganglion cell axons from the onset of neurite elongation at E3 and is limited to the OFL until E10. The intensity of anti-MAP5 staining in the OFL and optic nerve decreases after E7, which corresponds with a decrease in the number of actively growing ganglion cell axons. By E14, anti-MAP5 stains five layers in the IPL that correspond with layers of amacrine cell process arborizations. Western blots of E10 brain microtubule proteins show that MAP2 is represented by both a 260 x 10(3) Mr protein and a 60-65 x 10(3) Mr protein; the latter is much more abundant. Anti-MAP5 recognizes a 320 x 10(3) Mr brain microtubule protein in both the quail and the rat. We conclude that the cellular distribution, developmental regulation and molecular forms of MAP2 and MAP5 are similar in the rat and quail, suggesting that these molecules have conserved and hence fundamental roles in the growth and differentiation of neuronal processes.     

A 70-Kilodalton Microtubule-Associated Protein (MAP2c), Related to MAP2

Microtubule-associated protein 2 (MAP2) from adult brain consists of a pair of high molecular mass (280 kilodaltons) polypeptides, MAP2a and MAP2b. Juvenile brain microtubules also contain a 70-kilodalton protein that cross-reacts with monoclonal antibodies against these high molecular weight MAP2s. We have analyzed the relationship between this 70-kilodalton protein and MAP2 by peptide mapping. Our results show that the 70-kilodalton species bears strong homology to the MAP2 molecules and that it is distinct from the tau MAPs. We propose the name MAP2c for this low molecular weight MAP2 species. MAP2c is developmentally regulated in brain, being more abundant in neonatal tissue than in the adult. In several cell lines, MAP2c is the sole MAP2 species expressed. We examined homogenates from both juvenile brain and MAP2c-containing cell lines for evidence of a protease activity that might be responsible for generating MAP2c from either MAP2a or MAP2b. No such activity was found, suggesting that MAP2c is an independently synthesized MAP2 species some 200 kilodaltons smaller than the previously recognized forms.     

膳食锌对小鼠脑组织微管相关蛋白2表达的影响

本工作观察了膳食锌与脑组织微管相关蛋白２（ＭＡＰ２）之间的联系,并探讨了微量元素锌调节微管聚合作用的可能机制。ＩＣＲ初孕小鼠８０ 孕期和哺乳局喂不同锌水平饲料,随机分为５组：严重缺锌组,轻度缺锌组,轻度缺锌组,适锌组,高锌对喂组及高锌组,它们饲料的锌水平分别为１,５,３０,１００和１００ｍｇ／ｋｇ。     

MAP2a, an Alternatively Spliced Variant of Microtubule-Associated Protein 2

Abstract: MAP2, a dendritically localized microtubule-associated protein (MAP), consists of a pair of high molecular mass (280 kDa) polypeptides, MAP2a and MAP2b, and several low molecular mass (70 kDa) proteins called MAP2c. Although MAP2b and MAP2c have been shown to arise via alternative splicing, it was not clear whether MAP2a is also created by alternative splicing or by posttranslational modification. Using epitope peptide mapping, we have demonstrated that an element specific to MAP2a is situated at its N-terminal end. A cDNA clone from an adult rat brain library was found to contain an additional 246 nucleotides situated at the 5' end of the 9-kb MAP2 mRNA. Antibodies generated against the encoded protein sequence recognize specifically MAP2a in rat brain homogenates. Moreover, although MAP2a, like MAP2b, is found in dendrites and cell bodies, its temporal appearance and cell type-specific distribution in rat brain differs from MAP2b.     

Naturally occurring human autoantibodies recognize a fetal brain antigen identified as microtubulus associated protein 1B

We have recently reported naturally occurring autoantibodies against a large fetal brain antigen (FBA). Now we describe the process of purification and identification of this particular FBA. The brains of newborn rabbits were solubilized and purified with preparative gel electrophoresis. The protein fractions were concentrated and desalted and the fractions were tested by a known positive serum. On membrane digestion of the FBA-band gave a twelve amino acid sequence that resulted in best identity score for mouse, rat and human microtubule-associated protein (MAP) 1B: a member of the microtubule-associated protein family. Monoclonal anti-MAP1B recognized a band in immunoblots of the brain homogenate and of the partially purified fractions with the same electrophoretic mobility as that recognized by a known anti-FBA positive serum. When adult rabbit brain was used as an antigen, the anti-MAP1B failed to recognize any bands on immunoblots. MAP lB has not been previously known as an autoantigen, even though many structural proteins of the neuronal cytoskeleton are known to be targets of naturally occurring autoantibodies. MAP 1B is a functionally important regulatory protein in the developing brain; thus autoantibodies against MAP1B may affect the normal development.     

Evidence for expression of some microtubule-associated protein 1B in neurons as a plasma membrane glycoprotein

Microtubule-associated protein (MAP) 1B is a high-molecular-weight cytoskeletal protein that is abundant in developing neuronal processes and appears to be necessary for axonal growth. Various biochemical and immunocytochemical results are reported, indicating that a significant fraction of MAP1B is expressed as an integral membrane glycoprotein in vesicles and the plasma membrane of neurons. MAP1B is present in microsomal fractions isolated from developing rat brain and fractionates across a sucrose gradient in a manner similar to synaptophysin, a well-known vesicular and plasma membrane protein. MAP1B is also in axolemma-enriched fractions (AEFs) isolated from myelinated axons of rat brain. MAP1B in AEFs and membrane fractions from cultured dorsal root ganglion neurons (DRGNs) remains membrane-associated following high-salt washes and contains sialic acid. Furthermore, MAP1B in intact DRGNs is readily degraded by extracellular trypsin and is labeled by the cell surface probe sulfosuccinimidobiotin. Immunocytochemical examination of DRGNs shows that MAP1B is concentrated in vesicle-rich varicosities along the length of axons. Myelinated peripheral nerves immunostained for MAP1B show an enrichment at the axonal plasma membrane. These observations demonstrate that some of the MAP1B in developing neurons is an integral plasma membrane glycoprotein.     

A microtubule-associated protein (MAP1) which is expressed at elevated levels during development of the rat cerebellum.

Monoclonal antibody (MAb) G10 labels a single high mol. wt. (HMW) band on Western blots of microtubule preparations from 2 day old rat brain. The G10 antigen is thermolabile and co-migrates with microtubule-associated protein (MAP)1 from young rat brain on low percentage (5%) polyacrylamide-SDS gels. The G10 antigen decreases by about five times from birth to adulthood in the rat cerebellum. The same single band is labelled on Western blots of homogenates of whole neonatal rat brain but no labelling is found using neonatal or adult kidney, lung or liver. We have therefore identified a brain-specific MAP1, designated MAP1(x). Immunofluorescence microscopy using MAb G10 on parasagittal sections of rat cerebella shows labelling of the newly formed molecular layer in 6 day old rats. Only a narrow band close to the pial surface is labelled in 18 day old animals, which disappears in the adult. Labelling of the cerebellar white matter found in young rats also disappears. Neurones but not flat cells in cerebellar cultures label with MAb G10. All staining patterns are consistent with an axonal distribution of the antigen. MAP1(x) may be part of a developmentally regulated microtubule structure.     

Heterogeneity in the Phosphorylation of Micro tubule-Associated Protein MAP 1B During Rat Brair Development

Abstract: The patterns of isoforms and of immunoreactivity of the microtubule-associated protein MAP1 B toward a panel of antibodies to phosphorylation-sensitive epitopes are different in distinct rat brain regions and change during development. This suggests the occurrence of a considerable degree of heterogeneity in the phosphorylation state of rat brain MAP1 B. It appears that MAP1 B can be phosphorylated at multiple sites that may be conventionally classified into at least two modes of phosphorylation. Mode I of phosphorylation induces significant upward shifts in the electrophoretic mobility of the protein, giving rise to "high" MAP1B isoforms, whereas the mode II of MAP1B phosphorylation does not greatly affect the electrophoretic mobility of the protein. These MAP1B phosphorylation modes are differentially regulated throughout development and show some regional specificity. Cytosolic MAP1 B is highly phosphorylated both at mode I and mode II sites in the developing rat brain, as well as in the adult olfactory bulb, where axonal growth takes place. In most adult rat brain regions, cytosolic MAP1B is highly phosphorylated at mode II sites but largely dephosphorylated at certain mode I sites. However, MAP1 B present in the particulate fraction of most rat brain region homogenates may be partially dephosphorylated at certain mode II sites, although it contains some phosphorylated mode I sites. These data are compatible with the view that different protein kinases, possibly including casein kinase II and proline-directed protein kinases, might regulate the state of phosphorylation of MAP1B in distinct localizations along the development of different neuronal populations in the brain.     

The juvenile microtubule-associated protein MAP2c is a rod-like molecule that forms antiparallel dimers.

We have developed a procedure to isolate the microtubule-associated protein 2c (MAP2c), a juvenile form of MAP2 occurring in mammalian brain. The shape, size, self-association, and antibody interactions of MAP2c were studied. Monomeric MAP2c is an elongated molecule with a length approximately 48 nm, considerably shorter than the higher molecular weight forms MAP2a or b of adult brain. Two monoclonal antibodies whose epitopes are near the N or C terminus, respectively, are located close to the opposite ends of the MAP2c rods. This places constraints on the types of internal folding of the molecule. MAP2c self-associates into dimers and fibrous aggregates. The dimers are predominantly antiparallel and nearly in register, as judged by antibody labeling.     

Molecular structure of microtubule-associated protein 2b and 2c from rat brain

Full length cDNA clones encoding microtubule-associated proteins (MAP) 2b and 2c from rat brain have been isolated and sequenced. The cDNA fragments spanning the coding regions for both MAP2b and MAP2c were assembled and expressed in Escherichia coli. The mobility of these bacterial expressed proteins in sodium dodecyl sulfate gels is identical to that of MAP2b and MAP2c from rat brain. The protein sequence of rat MAP2b has been compared to the full length sequence from mouse and the partial sequence from human high molecular weight MAP2. This comparison has revealed that MAP2b is composed of several highly conserved domains flanked by domains with extensive sequence divergence. Two of the conserved domains, found either at the NH2 or COOH terminus, overlap with the binding domain for the regulatory subunit of the cAMP-dependent protein kinase II and the microtubule-binding domain, respectively. A third homologous domain of unknown function lies in a central region of MAP2b. Secondary structure prediction suggests that the portion of MAP2b which extends from the microtubule surface is composed of an extensive number of alpha-helices separated by small turns which may account for the extended yet flexible structure of MAP2. Interestingly, the 4000-base pair deletion from the middle of MAP2b which generates MAP2c not only removes these helices, but also this third highly conserved MAP2b domain.     

Microtubule-associated proteins and the determination of neuronal form

1. The assembly of microtubules is essential for the maintenance of both the extension and the radial symmetry of axons and dendrites. Microtubule-associated proteins (MAPs) are implicated in this function because they promote tubulin polymerization and because they appear to be involved in cross-linking microtubules in the neuritic cytoplasm. 2. In a variety of species high molecular weight MAP2 is found only in dendrites and MAP tau is found only is axons, indicating that certain MAPs are associated with specific aspects of neuronal morphology. 3. All neuronal MAPs that have been studied are under strong developmental regulation with either their form or abundance changing between developing and adult brain. In both rat and Xenopus the change from "early" to "late" MAP forms occurs concurrently with the cessation of axon and dendrite growth and the maturation of neuronal morphology. 4. In situations where neuronal growth persists in the adult, such as retinal photoreceptor cells and the olfactory system, "early" MAPs continue to be expressed in the adult brain. 5. These results implicate MAPs in neuronal morphogenesis and suggest that "early" MAPs are involved in axon and dendrite growth whereas the "late" MAPs are involved in the stabilization of their mature form.     

Alzheimer's Disease Neurofibrillary Tangles Contain Mitosis-Specific Phosphoepitopes

Abstract: Paired helical filaments (PHFs) are the major components of neurofibrillary lesions present in Alzheimer's disease (AD). PHFs are composed of the microtubule-associated protein (MAP) τ, which is abnormally phosphorylated in AD. Normal fetal τ is also phosphorylated and shares certain phosphoepitopes with PHF-τ. The abnormal phosphorylation of PHF-τ is considered to be involved in the formation of PHFs and subsequent degeneration of AD neurons. We have previously shown that other neuronal MAPs, such as MAP1B, contain mitosis-specific phosphoepitopes. In addition to mitotic cells, these epitopes are also expressed in fetal brain and PC12 cells during differentiation and neurite outgrowth. One hypothesis regarding the etiology of AD involves the reactivation of a fetal-like state and mitotic conditions in selected neurons. To determine if similar mitosis-associated phosphoepitopes appeared in AD, sections of hippocampal tissue were stained for immunoreactivity with antibodies recognizing both τ and mitotic phosphoepitopes. Both the MPM2 mitotic phosphoepitope antibody and the AT8 PHF-τ antibody stained neurofibrillary lesions and colocalized to pyramidal neurons in AD samples. In addition, PHFs isolated from an AD brain reacted with both antibodies. The MPM2 antibody specifically reacted with τ in the isolated PHF fraction but not normal adult τ. In addition, MPM2 failed to react with normal fetal or adult τ obtained from rat brains. The MPM2 antibody also recognized human MAP1B; however, MAP1B was not present in the PHF fraction. Our results indicate that MPM2 recognized a phosphoepitope present on PHF-τ. Because normal fetal or adult rat brain τ did not express the MPM2 epitope, it is likely that this phosphoepitope is specific for the disease state.     

Association of microtubule-associated protein 2 (MAP 2) with microtubules and intermediate filaments in cultured brain cells

The classification of MAP 2 as a microtubule-associated protein is based on its affinity for microtubules in vitro and its filamentous distribution in cultured cells. We sought to determine whether MAP 2 is also able to bind in situ to organelles other than microtubules. For this purpose, primary cultures of rat brain cells were stained for immunofluorescence microscopy with a rabbit anti-MAP 2 antibody prepared in our laboratory, as well as with antibodies to vimentin, an intermediate filament protein, and to tubulin, the major subunit of microtubules. MAP 2 was present on cytoplasmic fibers in neurons and in a subpopulation of the flat cells present in the cultures. Our observations were concentrated on the flat cells because of their suitability for high-resolution immunofluorescence microscopy. Double antibody staining revealed co-localization of MAP 2 with both tubulin and vimentin in the flat cells. Pretreatment of the cultures with vinblastine resulted in the redistribution of MAP 2 into perinuclear cables that contained vimentin. Tubulin paracrystals were not stained by anti-MAP 2. In cells extracted with digitonin, the normal fibrillar distribution of MAP 2 was resistant to several treatments (PIPES buffer plus 10 mM Ca++, phosphate buffer at pH 7 or 9) that induced depolymerization of microtubules, but not intermediate filaments. Staining of the primary brain cells was not observed with preimmune serum nor with immune serum adsorbed prior to use with pure MAP 2. We detected MAP 2 on intermediate filaments not only with anti-MAP 2 serum, but also with affinity purified anti-MAP 2 and with a monoclonal anti-MAP 2 prepared in another laboratory. We conclude from these experiments that material recognized by anti-MAP 2 antibodies associates with both microtubules and intermediate filaments. We propose that one function of MAP 2 is to cross-link the two types of cellular filaments.